The Discovery and Early Days of TGF-ß: A Historical Perspective.

Transforming growth factors (TGFs) were discovered as activities that were secreted by cancer cells, and later by normal cells, and had the ability to phenotypically and reversibly transform immortalized fibroblasts. TGF-ß distinguished itself from TGF-α because it did not bind to the same epidermal growth factor (EGF) receptor as TGF-α and, therefore, acted through different cell-surface receptors and signaling mediators. This review summarizes the discovery of TGF-ß, the early developments in its molecular and biological characterization with its many biological activities in different cell and tissue contexts and its roles in disease, the realization that there is a family of secreted TGF-ß-related proteins with many differentiation functions in development and activities in normal cell and tissue physiology, and the subsequent identification and characterization of the receptors and effectors that mediate TGF-ß family signaling responses.

Transient tumor-fibroblast interactions increase tumor cell malignancy by a TGF-Beta mediated mechanism in a mouse xenograft model of breast cancer.

Carcinoma are complex societies of mutually interacting cells in which there is a progressive failure of normal homeostatic mechanisms, causing the parenchymal component to expand inappropriately and ultimately to disseminate to distant sites. When a cancer cell metastasizes, it first will be exposed to cancer associated fibroblasts in the immediate tumor microenvironment and then to normal fibroblasts as it traverses the underlying connective tissue towards the bloodstream. The interaction of tumor cells with stromal fibroblasts influences tumor biology by mechanisms that are not yet fully understood. Here, we report a role for normal stroma fibroblasts in the progression of invasive tumors to metastatic tumors. Using a coculture system of human metastatic breast cancer cells (MCF10CA1a) and normal murine dermal fibroblasts, we found that medium conditioned by cocultures of the two cell types (CoCM) increased migration and scattering of MCF10CA1a cells in vitro, whereas medium conditioned by homotypic cultures had little effect. Transient treatment of MCF10CA1a cells with CoCM in vitro accelerated tumor growth at orthotopic sites in vivo, and resulted in an expanded pattern of metastatic engraftment. The effects of CoCM on MCF10CA1a cells were dependent on small amounts of active TGF-beta1 secreted by fibroblasts under the influence of the tumor cells, and required intact ALK5-, p38-, and JNK signaling in the tumor cells. In conclusion, these results demonstrate that transient interactions between tumor cells and normal fibroblasts can modify the acellular component of the local microenvironment such that it induces long-lasting increases in tumorigenicity and alters the metastatic pattern of the cancer cells in vivo. TGF-beta appears to be a key player in this process, providing further rationale for the development of anti-cancer therapeutics that target the TGF-beta pathway.

The type I BMP receptors, Bmpr1a and Acvr1, activate multiple signaling pathways to regulate lens formation.

BMPs play multiple roles in development and BMP signaling is essential for lens formation. However, the mechanisms by which BMP receptors function in vertebrate development are incompletely understood. To determine the downstream effectors of BMP signaling and their functions in the ectoderm that will form the lens, we deleted the genes encoding the type I BMP receptors, Bmpr1a and Acvr1, and the canonical transducers of BMP signaling, Smad4, Smad1 and Smad5. Bmpr1a and Acvr1 regulated cell survival and proliferation, respectively. Absence of both receptors interfered with the expression of proteins involved in normal lens development and prevented lens formation, demonstrating that BMPs induce lens formation by acting directly on the prospective lens ectoderm. Remarkably, the canonical Smad signaling pathway was not needed for most of these processes. Lens formation, placode cell proliferation, the expression of FoxE3, a lens-specific transcription factor, and the lens protein, alphaA-crystallin were regulated by BMP receptors in a Smad-independent manner. Placode cell survival was promoted by R-Smad signaling, but in a manner that did not involve Smad4. Of the responses tested, only maintaining a high level of Sox2 protein, a transcription factor expressed early in placode formation, required the canonical Smad pathway. A key function of Smad-independent BMP receptor signaling may be reorganization of actin cytoskeleton to drive lens invagination.

FGF-regulated BMP signaling is required for eyelid closure and to specify conjunctival epithelial cell fate.

There are conflicting reports about whether BMP signaling is required for eyelid closure during fetal development. This question was addressed using mice deficient in BMP or TGFbeta signaling in prospective eyelid and conjunctival epithelial cells. Genes encoding two type I BMP receptors, the type II TGFbeta receptor, two BMP- or two TGFbeta-activated R-Smads or the co-Smad Smad4 were deleted from the ocular surface ectoderm using Cre recombinase. Only mice with deletion of components of the BMP pathway had an 'eyelid open at birth' phenotype. Mice lacking Fgf10 or Fgfr2 also have open eyelids at birth. To better understand the pathways that regulate BMP expression and function during eyelid development, we localized BMPs and BMP signaling intermediates in Fgfr2 and Smad4 conditional knockout (CKO) mice. We found that Fgfr2 was required for the expression of Bmp4, the normal distribution of Shh signaling and for preserving the differentiation of the conjunctival epithelium. FGF signaling also promoted the expression of the Wnt antagonist Sfrp1 and suppressed Wnt signaling in the prospective eyelid epithelial cells, independently of BMP function. Transcripts encoding Foxc1 and Foxc2, which were previously shown to be necessary for eyelid closure, were not detectable in Smad4(CKO) animals. c-Jun, another key regulator of eyelid closure, was present and phosphorylated in eyelid periderm cells at the time of fusion, but failed to translocate to the nucleus in the absence of BMP function. Smad4(CKO) mice also showed premature differentiation of the conjunctival epithelium, conjunctival hyperplasia and the acquisition of epidermal characteristics, including formation of an ectopic row of hair follicles in place of the Meibomian glands. A second row of eyelashes is a feature of human lymphedema-distichiasis syndrome, which is associated with mutations in FOXC2.

Transforming growth factor-beta/Smad3 signaling regulates insulin gene transcription and pancreatic islet beta-cell function.

Pancreatic islet beta-cell dysfunction is a signature feature of Type 2 diabetes pathogenesis. Consequently, knowledge of signals that regulate beta-cell function is of immense clinical relevance. Transforming growth factor (TGF)-beta signaling plays a critical role in pancreatic development although the role of this pathway in the adult pancreas is obscure. Here, we define an important role of the TGF-beta pathway in regulation of insulin gene transcription and beta-cell function. We identify insulin as a TGF-beta target gene and show that the TGF-beta signaling effector Smad3 occupies the insulin gene promoter and represses insulin gene transcription. In contrast, Smad3 small interfering RNAs relieve insulin transcriptional repression and enhance insulin levels. Transduction of adenoviral Smad3 into primary human and non-human primate islets suppresses insulin content, whereas, dominant-negative Smad3 enhances insulin levels. Consistent with this, Smad3-deficient mice exhibit moderate hyperinsulinemia and mild hypoglycemia. Moreover, Smad3 deficiency results in improved glucose tolerance and enhanced glucose-stimulated insulin secretion in vivo. In ex vivo perifusion assays, Smad3-deficient islets exhibit improved glucose-stimulated insulin release. Interestingly, Smad3-deficient islets harbor an activated insulin-receptor signaling pathway and TGF-beta signaling regulates expression of genes involved in beta-cell function. Together, these studies emphasize TGF-beta/Smad3 signaling as an important regulator of insulin gene transcription and beta-cell function and suggest that components of the TGF-beta signaling pathway may be dysregulated in diabetes.

Acute wounds accelerate tumorigenesis by a T cell-dependent mechanism.

We investigated the influence of acute wounding on tumor growth in a syngeneic mouse breast cancer model. Metastatic mouse breast cancer cells (4T1) were orthotopically injected into the mammary fat pads of BALB/c mice, and animals were wounded locally by full thickness dermal incisions above the mammary fat pads or remotely above the scapula 9 days later. Local, but not remote, wounding increased tumor size when compared with sham treatment. Injection of wound fluid close to the tumor site increased tumor growth, whereas in vitro wound fluid compared with serum increased the proliferation rate of 4T1 cells. Our results show that wound stroma can unfavorably influence growth of nearby tumors. This effect is T cell-dependent, as local wounding had no effect on tumor growth in nu/nu mice. The effect of wounding on tumor growth can be mimicked by acellular wound fluid, suggesting that T cells secrete or mediate secretion of cytokines or growth factors that then accelerate tumor growth. Here, we define an experimental model of wound-promoted tumor growth that will enable us to identify mechanisms and therapeutic targets to reduce the negative effect of tissue repair on residual tumors.

Functions of the type 1 BMP receptor Acvr1 (Alk2) in lens development: cell proliferation, terminal differentiation, and survival.

PURPOSE: Bone morphogenetic protein (BMP) signaling is essential for the induction and subsequent development of the lens. The purpose of this study was to analyze the function(s) of the type 1 BMP receptor, Acvr1, in lens development. METHODS: Acvr1 was deleted from the surface ectoderm of mouse embryos on embryonic day 9 using the Cre-loxP METHOD: Cell proliferation, cell cycle exit, and apoptosis were measured in tissue sections by immunohistochemistry, immunofluorescence, and TUNEL staining. RESULTS: Lenses formed in the absence of Acvr1. However, Acvr1(CKO) (conditional knockout) lenses were small. Acvr1 signaling promoted proliferation at early stages of lens formation but inhibited proliferation at later stages. Inhibition of cell proliferation by Acvr1 was necessary for the proper regionalization of the lens epithelium and promoted the withdrawal of lens fiber cells from the cell cycle. In spite of the failure of all Acvr1(CKO) fiber cells to withdraw from the cell cycle, they expressed proteins characteristic of differentiated fiber cells. Although the stimulation of proliferation was Smad independent, the ability of Acvr1 to promote cell cycle exit later in development depended on classical R-Smad-Smad4 signaling. Loss of Acvr1 led to an increase in apoptosis of lens epithelial and fiber cells. Increased cell death, together with the initial decrease in proliferation, appeared to account for the smaller sizes of the Acvr1(CKO) lenses. CONCLUSIONS: This study revealed a novel switch in the functions of Acvr1 in regulating lens cell proliferation. Previously unknown functions mediated by this receptor included regionalization of the lens epithelium and cell cycle exit during fiber cell differentiation.

Absence of Smad3 induces neutrophil migration after cutaneous irradiation: possible contribution to subsequent radioprotection.

Our previous work showed that 6 weeks after cutaneous irradiation, mice null (knockout, KO) for Smad3, a cytoplasmic downstream mediator of transforming growth factor-beta, demonstrate less epidermal acanthosis and dermal inflammation than wild-type (WT) Smad3 mice. Analysis of the kinetics of inflammation showed that 6 to 8 hours after skin irradiation, there was a transient sevenfold increase in neutrophil influx in Smad3 KO mice compared with WT. Herein we describe bone marrow transplantation and skin grafting between WT and KO mice to assess the contribution of the neutrophil genotype compared with that of irradiated skin to the induction of neutrophil migration after irradiation. Results from bone marrow transplantation showed that WT marrow transplanted into KO mice enhanced neutrophil migration 6 to 8 hours after irradiation by 3.2-fold compared with KO marrow in WT mice. KO skin grafted onto either WT or KO animals showed a sixfold elevation of neutrophils after irradiation compared with grafted WT skin. These results suggest that the genotype of the irradiated skin, rather than the inflammatory cell, controls neutrophil influx. Circulating neutrophils, increased in WT mice after injection of granulocyte colony-stimulating factor, resulted in increased neutrophil migration to the skin 6 to 8 hours after irradiation and less skin damage 6 weeks after irradiation compared with untreated WT mice. Thus, early responses, including enhanced neutrophil influx, appear to contribute to subsequent cutaneous radioprotection.

Lentiviral reporter constructs for fluorescence tracking of the temporospatial pattern of Smad3 signaling.

Canonical TGF-beta is involved in cell differentiation, tissue maintenance, and wound healing, but also plays a central role in the pathogenesis of diseases such as cancer Here we describe a lentivirus-based reporter vector system expressing green fluorescent protein (GFP) or red fluorescent protein (RFP) under the control of a Smad3-responsive element (CAGA)12 that allows observation of the temporospatial pattern of endogeneous Smad3-mediated signaling on a cellular level. Use of this method will be valuable to identify cells with active Smad3 signaling and investigate the role of endogenous Smad3 signaling in complex systems such as co-cultures in vitro, or in tumors during tumor cell invasion and metastasis in vivo.

Absence of Smad3 confers radioprotection through modulation of ERK-MAPK in primary dermal fibroblasts.

BACKGROUND: Transforming growth factor-beta1 (TGF-beta1), a key biological mediator following ionizing radiation, plays a role in a complex tissue reaction involved in local radiation-induced pathological damage. Knocking out Smad3 (S3KO), a downstream signaling intermediate in the TGF-beta pathway, in mice protects their skin from radiation damage as demonstrated by decreased epithelial acanthosis and dermal fibrosis as compared to Smad3 wild-type (S3WT) mice. OBJECTIVE: The present study was designed to investigate the molecular mechanisms contributing to increased radioprotection in the absence of Smad3. METHODS: Primary dermal fibroblasts derived from S3WT and KO mice were exposed to 5Gy ionizing radiation in vitro. Western blot analyses, immunocytochemistry, and reporter transfections were used to dissect the radiation-induced events. RESULTS: There was increased phosphorylation of ERK-MAPK, p53 and H2A.X in S3KO compared to the S3WT fibroblasts, implicating them in a key signaling cascade in response of these cells to radiation. Pro-fibrotic gene expression was decreased in S3KO fibroblasts post-irradiation. CONCLUSION: The absence of Smad3 may decrease radio-responsiveness by increasing activation of DNA damage sensing mechanisms and decreasing induction of pro-fibrotic genes.

Endothelial-to-mesenchymal transition contributes to cardiac fibrosis.

Cardiac fibrosis, associated with a decreased extent of microvasculature and with disruption of normal myocardial structures, results from excessive deposition of extracellular matrix, which is mediated by the recruitment of fibroblasts. The source of these fibroblasts is unclear and specific anti-fibrotic therapies are not currently available. Here we show that cardiac fibrosis is associated with the emergence of fibroblasts originating from endothelial cells, suggesting an endothelial-mesenchymal transition (EndMT) similar to events that occur during formation of the atrioventricular cushion in the embryonic heart. Transforming growth factor-beta1 (TGF-beta1) induced endothelial cells to undergo EndMT, whereas bone morphogenic protein 7 (BMP-7) preserved the endothelial phenotype. The systemic administration of recombinant human BMP-7 (rhBMP-7) significantly inhibited EndMT and the progression of cardiac fibrosis in mouse models of pressure overload and chronic allograft rejection. Our findings show that EndMT contributes to the progression of cardiac fibrosis and that rhBMP-7 can be used to inhibit EndMT and to intervene in the progression of chronic heart disease associated with fibrosis.

SNIP1 is a candidate modifier of the transcriptional activity of c-Myc on E box-dependent target genes.

Using a yeast two-hybrid screen, we found that SNIP1 (Smad nuclear-interacting protein 1) associates with c-Myc, a key regulator of cell proliferation and transformation. We demonstrate that SNIP1 functions as an important regulator of c-Myc activity, binding the N terminus of c-Myc through its own C terminus, and that SNIP1 enhances the transcriptional activity of c-Myc both by stabilizing it against proteosomal degradation and by bridging the c-Myc/p300 complex. These effects of SNIP1 on c-Myc likely contribute to synergistic effects of SNIP1, c-Myc, and H-Ras in inducing formation of foci in an in vitro transformation assay and also in supporting anchorage-independent growth. The significant association of SNIP1 and c-Myc staining in a non-small cell lung cancer tissue array is further evidence that their activities might be linked and suggests that SNIP1 might be an important modulator of c-Myc activity in carcinogenesis.

Smad3 deficiency alters key structural elements of the extracellular matrix and mechanotransduction of wound closure.

The loss of TGFbeta or its downstream mediator, Smad3, key players in tissue repair, accelerates closure of incisional wounds in mice. In contrast, we now report that excisional ear wounds in mice lacking Smad3 enlarge compared with wild-type controls resulting from changes in extracellular matrix molecules, which alter the mechanotransduction properties of these wounds. Specifically, levels of elastin and glycosoaminoglycans are increased, collagen fibers are more compactly organized, and matrix modulators like integrins, TGFbeta1, and matrix metalloproteinases (MMPs) are altered both basally and after wounding in Smad3 knockout mice. Mechanical testing of dorsal skin correlates these changes in matrix composition with functional parameters, specifically an increased elastic modulus, suggesting an imbalance of tissue forces. We propose that the altered mechanical elastic properties translate into a persistent retractile force that is opposed by decreased wound contractile forces contributing to the enlarging ear wound in Smad3 knockout mice. These studies highlight a previously undescribed role for Smad3 in the mechanotransduction of matrix unsupported ear wound closure.

Smad4-expression is decreased in breast cancer tissues: a retrospective study.

BACKGROUND: Although transforming growth factor beta (TGF-beta) typically inhibits proliferation of epithelial cells, consistent with a tumor suppressor activity, it paradoxically also exhibits pro-metastatic activity in the later stages of carcinogenesis. Since tumors often display altered TGF-beta signaling, particularly involving the Smad-pathway, we investigated the role of Smad4-expression in breast cancer. METHODS: Smad4 expression was investigated by immunohistochemistry in formalin-fixed, paraffin-embedded tissue from 197 samples of primary breast cancer obtained between 1986 and 1998. The prognostic value of Smad4-expression was analyzed. RESULTS: Smad4 expression was found to be reduced in lobular and ductal breast carcinoma as compared to surrounding uninvolved lobular and ductal breast epithelia (p < 0.001, n = 50). Smad4-expression correlated positively with expression of TGF-beta-receptor I (p < 0.001, n = 197) and TGF-beta-receptor II (p < 0.001, n = 197), but showed no significant correlation with tumor size, metastases, nodal status, histological grade, histological type, or estrogen receptor expression. While not achieving statistical significance, there was a trend towards longer survival times in patients with Smad4 negative tumors. CONCLUSION: According to the suggested role of Smad4 as a tumor suppressor we observed that expression of Smad4 is lower in human breast cancer than in surrounding breast epithelium. However, we also observed a trend towards longer survival times in Smad4-negative patients, indicating the complex role of TGF-beta signaling in tumor progression.

Smad3 is key to TGF-beta-mediated epithelial-to-mesenchymal transition, fibrosis, tumor suppression and metastasis.

Smads2 and 3 transduce signals of TGF-beta from the cell surface to the nucleus. We used mice with a targeted deletion of Smad3 to study the specific contributions of this signaling pathway to pathogenic effects of TGF-beta. Focusing on models involving epithelial-to-mesenchymal transition (EMT), including injury to the lens and retina of the eye and to the kidney, we have found that loss of Smad3 blocks EMT and attenuates development of fibrotic sequelae. Smad3 also plays a critical role in both the tumor suppressor and pro-metastatic effects of TGF-beta in carcinogenesis. These observations suggest that development of small molecule inhibitors of Smad3 might have clinical application in treatment of fibrotic diseases as well as late stage cancers.

Inhibition of p38MAP kinase suppresses fibrotic reaction of retinal pigment epithelial cells.

Proliferative vitreoretinopathy (PVR) is one of the major causes of the failure of retinal detachment surgery. Its pathogenesis includes a fibrotic reaction by the retinal pigment epithelium and other retina-derived non-neural cells, leading to fixation of the detached retina. We examined the role of p38 mitogen-activated protein kinase (MAPK) in transforming growth factor (TGF)-beta2-dependent enhancement of the fibrogenic reaction in a human retinal pigment epithelial cell line, ARPE-19, and also evaluated the therapeutic efficacy of inhibiting p38MAPK by adenoviral gene transfer of dominant-negative (DN) p38MAPK in a mouse model of PVR. Exogenous TGF-beta2 activates p38MAPK in ARPE-19 cells. It also suppresses cell proliferation, but this was unaffected by addition of the p38MAPK inhibitor, SB202190. SB202190 interfered with TGF-beta2-dependent cell migration and production of collagen type I and fibronectin, but had no effect on basal levels of these activities. While SB202190 did not affect phosphorylation of the C-terminus of Smads2/3, it did suppress the transcriptional activity of Smads3/4 as indicated by a reporter gene, CAGA12-Luc. Gene transfer of DN-p38MAPK attenuated the post-retinal detachment fibrotic reaction of the retinal pigment epithelium in vivo in mice, supporting its effectiveness in preventing/treating PVR.

Breast cancer cells induce stromal fibroblasts to express MMP-9 via secretion of TNF-alpha and TGF-beta.

We used 2D-cocultures employing fibroblasts of different genetic backgrounds and MCF10A-derived human breast epithelial cells of increasingly malignant potential to investigate tumor-stroma interactions in breast cancer and to identify possible signaling pathways involved. Tumor cells induced expression of matrix-metalloproteinase 9 (MMP-9) in fibroblasts in a pattern dependent on the degree of their malignancy. In-situ zymography localized the main gelatinolytic activity around stromal cells in cocultures and xenografted tumors. Use of Smad3 knockout fibroblasts, small molecule inhibitors, and neutralizing antibodies showed that MMP-9 expression was induced by tumor cell-derived TNF-alpha and TGF-beta, dependent on Smad-, Ras-, and PI3-kinase-signaling, and likewise modulated by subsequent HGF- and EGF-signaling. Together, our results indicate that MMP-9 levels in tumor fibroblasts are regulated by a complex tumor-stroma cross-talk, involving multiple ligands and cellular signaling pathways.

Expression of Smad7 in mouse eyes accelerates healing of corneal tissue after exposure to alkali.

Damage to the cornea from chemical burns is a serious clinical problem that often leads to permanent visual impairment. Because transforming growth factor (TGF)-beta has been implicated in the response to corneal injury, we evaluated the effects of altered TGF-beta signaling in a corneal alkali burn model using mice treated topically with an adenovirus (Ad) expressing inhibitory Smad7 and mice with a targeted deletion of the TGF-beta/activin signaling mediator Smad3. Expression of exogenous Smad7 in burned corneal tissue resulted in reduced activation of Smad signaling and nuclear factor-kappaB signaling via RelA/p65. Resurfacing of the burned cornea by conjunctival epithelium and its differentiation to cornea-like epithelium were both accelerated in Smad7-Ad-treated corneas with suppressed stromal ulceration, opacification, and neovascularization 20 days after injury. Introduction of the Smad7 gene suppressed invasion of monocytes/macrophages and expression of monocyte/macrophage chemotactic protein-1, TGF-beta1, TGF-beta2, vascular endothelial growth factor, matrix metalloproteinase-9, and tissue inhibitors of metalloproteinase-2 and abolished the generation of myofibroblasts. Although acceleration of healing of the burned cornea was also observed in mice lacking Smad3, the effects on epithelial and stromal healing were less pronounced than those in corneas treated with Smad7. Together these data suggest that overexpression of Smad7 may have effects beyond those of simply blocking Smad3/TGF-beta signaling and may represent an effective new strategy for treatment of ocular burns.

TGF-beta and vitamin D3 utilize distinct pathways to suppress IL-12 production and modulate rapid differentiation of human monocytes into CD83+ dendritic cells.

We previously demonstrated that agents known to signal infection or inflammation can rapidly and directly drive differentiation of human CD14+ monocytes into CD83+ dendritic cells (DCs) when introduced to cells under serum-free conditions. In this study, we evaluated the effects of TGF-beta and vitamin D3 (VitD3) on the proportion and function of monocytes that adopt DC characteristics. TGF-beta significantly decreased the proportion of cells that rapidly adopted stable DC characteristics in response to LPS, but had little or no effect on calcium ionophore-induced differentiation. In contrast, VitD3 showed no such pathway specificity and dramatically suppressed differentiation of monocytes into DCs in response to these agents. Both TGF-beta and VitD3 altered cytokine and chemokine production in LPS-treated monocytes, inhibited IL-12 and IL-10 secretion, and decreased the functional capacity of DCs. Despite the similar effects of TGF-beta and VitD3, there are significant differences in the signaling pathways used by these agents, as evidenced by their distinct effects on LPS- and calcium ionophore-induced DC differentiation, on LPS-induced secretion of IL-10, and on two members of the NF-kappaB family of transcription factors, RelB and cRel. These studies identify TGF-beta and VitD3 as potent regulatory factors that use distinct pathways to suppress both the differentiation of DCs as well as their capacity to secrete the Th1-polarizing cytokine IL-12. Because these agents are present in serum and negatively affect DC differentiation at physiological concentrations, our findings are likely to have significance regarding the in vivo role of TGF-beta and VitD3 in determining the type of immune responses.